Transfer RNA (tRNA) is an adapter molecule that decodes the information of mRNA (codon) and transfers the corresponding amino acid to a polypeptide chain being synthesized, and is a small-molecule RNA that plays a central role in protein translation. The anticodon loop of tRNA is chemically modified, which is necessary for the fidelity of translation. In particular, chemical modifications at the base at position 34, which is located in the anticodon, and the base at position 37, which is located in the vicinity of the anticodon, play an important role in controlling the accuracy of translation. In addition, it is believed that the breakdown of chemical modifications in the anticodon loop of tRNA is related to a disease.
Type 2 diabetes is a disease that occurs due to the combination of environmental factors and genetic factors. There are more than 200 million patients all over the world, and the number of patients is increasing in many countries. Type 2 diabetes is characterized by insulin resistance and/or abnormal insulin secretion in pancreatic β cells, but its main mechanism is still under discussion. Since 2007, large-scale genetic polymorphism epidemiological studies targeted at type 2 diabetes patients have been vigorously carried out around the world, and a large number of genetic single nucleotide polymorphisms (SNP) correlated with having diabetes have been identified. Among them, a large number of literatures have reported that SNPs of Cdkal1 (cdk5 regulatory associated protein 1-like 1) have the highest correlation with the onset of type 2 diabetes. It has also been revealed that a person carrying the risk allele of the Cdkal1 gene has poor glucose-responsive insulin secretion, but there is no correlation with obesity or insulin resistance. In addition, this genetic risk allele is common among Asians. Considering the phenotype of risk allele carriers, it is surmised that SNPs of Cdkal1 are involved in the onset of Asian type 2 diabetes.
Further, with respect to the physiological function of Cdkal1, it has been revealed that it is an enzyme that thiomethylates adenine37 in tRNA corresponding to lysine into 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) (Nonpatent Document 1: Arragain S., et al., J. Biol. Chem., 285, 28425-28433 (2010) and Nonpatent Document 2: Tomizawa et al., Uehara Kinen Seimeikagaku Zaidan Kenkyu Houkokusyo (The Research Reports of the Uehara Memorial Foundation), 25 (2011), Article No. 77). It has been shown that ms2t6A modification is necessary for accurate decoding of lysine codons (Nonpatent Document 1) and plays an important role in preventing the misreading of cognate codons, particularly in preventing misreading in the case where the translation rate is relatively high (Nonpatent Document 3: Tomizawa et al., Endocrine Journal 2011, 58 (10), 819-825).
It has been reported that in Cdkal1 knockout mice, mitochondria ATP production and first-phase insulin secretion are impaired. It has also been reported that pancreatic-β-cell-specific Cdkal1 knockout mice show the condition of type 2 diabetes, are observed to have pancreatic islet hypertrophy and impaired blood glucose control, and are hypersensitive to endoplasmic reticulum stress induced by high-fat diet (Nonpatent Document 3 and Nonpatent Document 4: Wei, F. Y. et al., J. Clin. Invest., 121, 3598-3608 (2011)).
That is, the present inventors have shown the following mechanism: when the expression or activity of Cdkal1 decreases due to genetic or environmental factors, the thiomethylation of lysine tRNA decreases, resulting in a decrease in the accuracy of insulin translation, which leads to the onset of type 2 diabetes. It has thus turned out that the thiomethylation of lysine tRNA is closely associated with the onset of diabetes. Meanwhile, thiomethylation is also present in mitochondrial tRNA encoded by mitochondrial DNA. It has been reported that the thiomethylation of mitochondrial tRNA is mediated by Cdk5rap1, and that a single nucleotide polymorphism mutation of the Cdk5rap1 gene is correlated with the onset of leukoplakia (Nonpatent Document 1 and Nonpatent Document 5: Reiter, V. et al., Nucleic Acids Res. 40, 6235-6240 (2012)). Like this, the thiomethylation of tRNA has been attracting attention as a new disease biomarker.
As a method for detecting the thiomethylation of tRNA, a detection method using a mass spectrometry method is generally used (Nonpatent Document 6: Suzuki, T. et al., Method. Enzymol., 425, 211-229 (2007)). In a mass spectrometry method, first, tRNA is purified from tissue or cells, and then tRNA is digested with nuclease into several oligonucleotides. Subsequently, the digested oligonucleotides are purified and analyzed by reversed-phase liquid chromatography and a mass spectrometer to detect thiomethylation. However, the detection of thiomethylation by mass spectrometry requires large amounts of RNA (in the unit of mg). Clinical samples are generally limited in amount, and it is difficult to obtain samples in the unit of mg. Accordingly, it is difficult to detect thiomethylation by mass spectrometry using clinical samples. In addition, the thiomethylation detection method by mass spectrometry requires a large number of pretreatments as described above. Therefore, there are problems in that it takes several days to detect thiomethylation, which not only makes it impossible to perform quick detection but also results in increased cost. Further, it is difficult to treat a large number of samples simultaneously. In addition, mass spectrometers are extremely expensive, and also the use thereof requires skills and experiences. Therefore, the analysis is difficult to perform in ordinary medical institutions or laboratories.
As described above, a mass spectrometry method takes time and cost, and further it is difficult to obtain large amounts of clinical samples. Because of these problems, a mass spectrometry method cannot be clinically applied as a method for detecting the thiomethylation of RNA. Therefore, there has been a demand for a novel detection method that is capable of detecting the thiomethylation of RNA efficiently, that is, quickly at low cost, and further allows for detection using a small amount of sample.